Histological and immunohistochemical features suggesting aetiological differences in lymph node and (muco)cutaneous feline tuberculosis lesions.

OBJECTIVES: To identify and describe histological and immunohistochemical criteria that may differentiate between skin and lymph node lesions associated with Mycobacterium (M.) bovis and M. microti in a diagnostic pathology setting. MATERIALS AND METHODS: Archived skin and lymph node biopsies of tuberculous lesions were stained with haematoxylin and eosin, Ziehl-Neelsen and Masson's Trichrome. Immunohistochemistry was performed to detect the expression of calprotectin, CD3 and Pax5. Samples were scored for histological parameters (i.e. granulomas with central necrosis versus small granulomas without central necrosis, percentage necrosis and/or multinucleated giant cells), number of acid-fast bacilli (bacterial index) and lesion percentage of fibrosis and positive immunohistochemical staining. RESULTS: Twenty-two samples were examined (M. bovis n=11, M. microti n=11). When controlling for age, gender and tissue, feline M. bovis-associated lesions more often featured large multi-layered granulomas with central necrosis. Conversely, this presentation was infrequent in feline M. microti-associated lesions, where small granulomas without central necrosis predominated. The presence of an outer fibrous capsule was variable in both groups, as was the bacterial index. There were no differences in intralesional expression of immunohistochemical markers. CLINICAL SIGNIFICANCE: Differences in the histological appearance of skin and lymph node lesions may help to infer feline infection with either M. bovis or M. microti at an earlier stage when investigating these cases, informing clinicians of the potential zoonotic risk. Importantly, cases of tuberculosis can present with numerous acid-fast bacilli. This implies that a high bacterial index does not infer infection with non-zoonotic non-tuberculous mycobacteria.

Quantitative analysis of ER alpha and GAD colocalization in the hippocampus of the adult female rat.

Despite the many effects of estrogen in the hippocampus, there has been little evidence that hippocampal principal cells express nuclear estrogen receptors. In the hippocampus, the alpha form of the nuclear estrogen receptor (ER alpha) has been localized to sparsely distributed cells with the morphological characteristics of inhibitory interneurons. Because inhibitory neurons may be involved in the effects of estrogen on hippocampal principal cells, quantitative description of ER alpha expression in gamma-aminobutyric acid (GABA)ergic (inhibitory) and non-GABAergic cells of the hippocampus is a key step in understanding the mechanism(s) of estrogen action on hippocampal circuitry. We used single and double-label immunohistochemistry for ER alpha and glutamic acid decarboxylase (GAD; a marker of GABAergic neurons) to determine the numbers and distributions of hippocampal GABAergic and non-GABAergic neurons that express ER alpha in the adult female rat. We found many more ER alpha-expressing cells in the hippocampus than any previous study and observed distinct dorsal vs. ventral differences in hippocampal ER alpha expression. In the dorsal hippocampus, most ER alpha-positive cells were also GAD positive; however, ER alpha was expressed in only a subset of GAD-positive cells. Double-labeled cells were concentrated at the border between str. radiatum and str. lacunosum-moleculare. In the ventral hippocampus, we found a very high number of ER alpha-positive cells, the majority of which were not immunoreactive for GAD and are likely to be pyramidal cells. These findings suggest that ER alpha can mediate the effects of estrogen primarily in GABAergic neurons in the dorsal hippocampus and in both GABAergic and non-GABAergic neurons in the ventral hippocampus.

Estrogen regulates functional inhibition of hippocampal CA1 pyramidal cells in the adult female rat.

Previous studies have focused considerable attention on the effects of estrogen on excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen increases the density of dendritic spines and synapses on CA1 pyramidal cells and increases the sensitivity of these cells to excitatory synaptic input. Little is known, however, about the effects of estrogen on inhibitory synaptic input to CA1 pyramidal cells. We have used immunohistochemistry for glutamic acid decarboxylase and whole-cell voltage-clamp recording of IPSCs and EPSCs at multiple time points after estrogen treatment to (1) investigate estrogen regulation of synaptic inhibition in CA1 and (2) evaluate how estrogen affects the interaction between inhibitory and excitatory input to CA1 pyramidal cells. We find that estrogen transiently suppresses GABA(A)-mediated inhibition of CA1 pyramidal cells at a time point before changes in excitatory input to these cells occur. This finding is consistent with the suggestion that transient disinhibition of CA1 pyramidal cells is involved in estrogen-induced dendritic spine formation. We have also found that at a later time after estrogen, inhibition of CA1 pyramidal cells recovers in parallel with enhancement of NMDA-mediated excitatory input. The concurrent enhancement of GABA(A) and NMDA-mediated input to CA1 pyramidal cells restores a balance of excitatory and inhibitory input to these cells and increases the potential dynamic range of CA1 pyramidal cell responses to synaptic input.

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: a serial electron-microscopic study.

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.

Effects of oestradiol on hippocampal circuitry.

Oestradiol produces structural and functional changes in hippocampal circuitry of adult female rats. The density of both dendritic spines and axospinous synapses on hippocampal CA1 pyramidal cells is regulated by oestradiol. Additionally, oestradiol-induced differences in synaptic connectivity are paralleled by changes in NMDA receptor binding, immunoreactivity for NMDA receptors and sensitivity to NMDA receptor-mediated synaptic input. Curiously, while oestradiol effects are observed in CA1 pyramidal cells, most evidence indicates that these cells lack genomic oestradiol receptors. In contrast, at least some inhibitory neurons in CA1 do express oestradiol receptors. Others' in vitro studies suggest that oestradiol-induced increases in spine density require an initial decrease in inhibitory (GABAergic) drive onto pyramidal cells. We have used single and double label immunohistochemistry for c-Fos (as a measure of neuronal activation) and glutamic acid decarboxylase 65 (as a marker for inhibitory circuitry) to determine: (1) which hippocampal neuronal populations are activated by oestradiol and the time-course of this activation, as well as (2) whether oestradiol affects inhibitory circuitry in vivo as it does in vitro. Our findings are consistent with the suggestion that oestradiol increases dendritic spine density through a mechanism involving disinhibition of pyramidal cells.

Kainic acid-induced mossy fiber sprouting and synapse formation in the dentate gyrus of rats.

In the kainic acid (KA) model of temporal lobe epilepsy, mossy fibers (MFs) are thought to establish recurrent excitatory synaptic contacts onto granule cells. This hypothesis was tested by intracellular labeling of granule cells with biocytin and identifying their synaptic contacts in the dentate molecular layer with electron microscopic (EM) techniques. Twenty-three granule cells from KA-treated animals and 14 granule cells from control rats were examined 2 to 4 months following KA at the light microscopic (LM) level; four cells showing MF sprouting were further characterized at the EM level. Timm staining revealed a time-dependent growth of aberrant MFs into the dentate inner molecular layer. The degree of sprouting was generally (but not invariably) correlated with the severity and frequency of seizures. LM examination of individual biocytin-labeled MF axon collaterals revealed enhanced collateralization and significantly increased numbers of synaptic MF boutons in the hilus compared to controls, as well as aberrant MF growth into the granule cell and molecular layers. EM examination of serially reconstructed, biocytin-labeled MF collaterals in the molecular layer revealed MF boutons that form asymmetrical synapses with dendritic shafts and spines of granule cells, including likely autaptic contacts on parent dendrites of the biocytin-labeled granule cell. These results constitute ultrastructural evidence for newly formed excitatory recurrent circuits, which might provide a structural basis for enhanced excitation and epileptogenesis in the hippocampus of KA-treated rats.

Estradiol induces a phasic Fos response in the hippocampal CA1 and CA3 regions of adult female rats.

Previous studies have shown that estradiol induces structural and functional changes in hippocampal CA1 pyramidal cells of the adult female rat. Estradiol increases the density of dendritic spines and axospinous synapses on CA1 pyramidal cells, and increases these cells' sensitivity to NMDA receptor-mediated synaptic input. Curiously, while estradiol effects are observed in CA1 pyramidal cells, the majority of the evidence indicates that these cells lack genomic estradiol receptors. In contrast, genomic estradiol receptors are expressed in at least some hippocampal interneurons in CA1. The goal of the present study was to determine which hippocampal neuronal populations are activated by estradiol, as determined by induction of c-Fos immunoreactivity, as well as the time-course of this activation. We quantified c-Fos expression in each of the major subdivisions of the hippocampus in adult female rats at various time points during the same estradiol treatment regimen known to regulate dendritic spines and synapses on CA1 pyramidal cells. Our results show a phasic estradiol-induced c-Fos response in the pyramidal cell layers of both CA1 and CA3. c-Fos was induced within 2 h of treatment, decreased at 6 and 12 h, and subsequently increased again at 24 h after treatment with estradiol. Double labeling for c-Fos and GAD 65 or GAD 67 suggests that c-Fos is induced primarily in principal cells, though a small proportion of GABAergic cells is also labeled. These estradiol-induced changes in c-Fos expression may reflect phasic neuronal activation and coupling to gene expression, which could be involved in estradiol's effects on excitatory synaptic connectivity in the hippocampus.

Estradiol facilitates kainic acid-induced, but not flurothyl-induced, behavioral seizure activity in adult female rats.

PURPOSE: This study was designed to determine whether previously demonstrated increases in hippocampal axospinous synapse density and NMDA receptor function induced by estradiol are paralleled by increased susceptibility to limbic (kainic acid induced) or generalized (flurothyl induced) behavioral seizures. METHODS: Kainic acid was injected systemically to ovariectomized adult female rats treated with either estradiol or oil vehicle. The latencies to each of five stages of seizure-related behaviors (staring, wet-dog shakes, head waving and chewing, forelimb clonus, rearing, and falling) were recorded for each animal. Flurothyl was administered by inhalation to ovariectomized adult female rats treated with estradiol alone, estradiol followed by short-term progesterone, or oil vehicle. The latencies to each of three stages of seizure-related behaviors (first myoclonic jerk, forelimb clonus, wild running and bouncing) were recorded for each animal. RESULTS: Estradiol treatment decreased the latency to seizure-related behaviors induced by kainic acid, but neither estradiol alone nor estradiol followed by progesterone had any effect on flurothyl-induced seizure-related behaviors. CONCLUSIONS: The same estradiol treatment paradigm known to induce structural and functional changes in the excitatory circuitry of the hippocampus facilitates the progression of kainic acid-induced seizures, which are known to involve the hippocampus, but has no effect on flurothyl-induced seizures. The lack of an effect of estradiol alone or estradiol followed by progesterone on flurothyl-induced seizures indicates that estradiol's effects on seizure susceptibility do not result from increased neuronal excitability throughout the brain, but rather involve action within the limbic system. The data suggest that structural and functional changes in hippocampal circuitry induced by estradiol may contribute to increased susceptibility to limbic seizure activity.

Effects of estrogen in the CNS.

Awareness of estrogen's effects on neural function is broadening rapidly. Areas of recent progress include increased understanding of estrogen signaling through both genomic and nongenomic pathways, as well as the mechanisms by which estrogen can induce or maintain synapses and protect neurons from a variety of insults. Findings in these areas demonstrate a role for estrogen that goes beyond direct control of reproductive function.

Electrophysiological and cellular effects of estrogen on neuronal function.

Estrogen exerts a variety of electrophysiological, neurotrophic, and metabolic effects on neurons in the adult central nervous system. Recent epidemiological studies have suggested that estrogen, as hormone replacement therapy in postmenopausal women, may be protective against Alzheimer's disease; the biological basis for a potential neuroprotective effect of estrogen in humans is an area of intense current research. This review summarizes electrophysiological and cellular effects of estrogen on neuronal function, with particular emphasis on hypothalamic and hippocampal neurons. Classic electrophysiological studies are compared with more recent cellular and molecular analyses in an effort to illuminate significant relationships between data gathered over the last 30 years and from varied sources. Hypotheses are made for the mechanisms of estrogen action in the brain as well as the functional consequences of estrogen's effects for both normal brain function and pathological states.

Estrogen-mediated structural and functional synaptic plasticity in the female rat hippocampus.

Light and electron microscopic studies have shown that ovarian steroids regulate the density and number of excitatory synaptic inputs to hippocampal pyramidal cells in the adult female rat; elevated levels of estradiol are associated with a higher density of dendritic spine synapses on CA1 pyramidal cells. Electrophysiological analyses indicate that these hormone-induced synapses increase hippocampal excitability as well as the potential for synaptic plasticity. Importantly, correlation of dendritic spine density and sensitivity to synaptic input of individual CA1 pyramidal cells from estradiol-treated and control animals suggests that synapses induced by estradiol may be a specialized subpopulation that contains primarily the NMDA subtype of glutamate receptor. The apparent NMDA receptor specificity of these synapses may be key to understanding their functional significance. Currently, the behavioral consequences of additional spine synapses are unknown. Numerous studies have aimed at correlating hormone-induced changes in hippocampal connectivity with differences in hippocampus-dependent spatial learning ability in mazes, but the results of these efforts have been equivocal. Anatomical, electrophysiological, and behavioral studies of estradiol-mediated hippocampal plasticity are reviewed. In conclusion, it is suggested that standard behavioral tests of hippocampal function are not sufficient to reveal the behavioral consequences of hormone-induced hippocampal plasticity. Rather, understanding the behavioral consequences of estradiol and progesterone effects on hippocampal connectivity may require analysis of the hippocampus' cognitive and spatial information processing functions in relation to alternative biologically relevant behaviors. A (nonexclusive) proposal that hormone-induced hippocampal plasticity may facilitate appropriate prepartum/maternal behavior is discussed.

Hormonal effects on the brain.

Changes in seizure frequency over the course of the menstrual cycle (i.e., catamenial epilepsy) have long been documented. Ovarian steroid hormones have a number of important short- and long-term effects on the brain that may contribute to this phenomenon. In particular, estrogen induces structural and functional changes in hippocampal neurons which may contribute significantly to increasing seizure susceptibility. This article reviews the mechanisms of action of steroid hormones on the basis of findings in animal models, with particular emphasis on the effects of estrogen on the hippocampus.

Estradiol increases the sensitivity of hippocampal CA1 pyramidal cells to NMDA receptor-mediated synaptic input: correlation with dendritic spine density.

Previous studies have shown that estradiol induces new dendritic spines and synapses on hippocampal CA1 pyramidal cells. We have assessed the consequences of estradiol-induced dendritic spines on CA1 pyramidal cell intrinsic and synaptic electrophysiological properties. Hippocampal slices were prepared from ovariectomized rats treated with either estradiol or oil vehicle. CA1 pyramidal cells were recorded and injected with biocytin to visualize spines. The association of dendritic spine density and electrophysiological parameters for each cell was then tested using linear regression analysis. We found a negative relationship between spine density and input resistance; however, no other intrinsic property measured was significantly associated with dendritic spine density. Glutamate receptor autoradiography demonstrated an estradiol-induced increase in binding to NMDA, but not AMPA, receptors. We then used input/output (I/O) curves (EPSP slope vs stimulus intensity) to determine whether the sensitivity of CA1 pyramidal cells to synaptic input is correlated with dendritic spine density. Consistent with the lack of an estradiol effect on AMPA receptor binding, we observed no relationship between the slope of an I/O curve generated under standard recording conditions, in which the AMPA receptor dominates the EPSP, and spine density. However, recording the pharmacologically isolated NMDA receptor-mediated component of the EPSP revealed a significant correlation between I/O slope and spine density. These results indicate that, in parallel with estradiol-induced increases in spine/synapse density and NMDA receptor binding, estradiol treatment increases sensitivity of CA1 pyramidal cells to NMDA receptor-mediated synaptic input; further, sensitivity to NMDA receptor-mediated synaptic input is well correlated with dendritic spine density.

Estradiol increases the frequency of multiple synapse boutons in the hippocampal CA1 region of the adult female rat.

The effect of estradiol to increase the density of dendritic spines and axospinous synapses on hippocampal CA1 pyramidal cells in the adult female rat has been well-documented. However, presynaptic involvement in this process of synapse elimination and formation in the adult is unknown. To address this issue, we have reconstructed 410 complete presynaptic boutons through coded serial electron micrographs of CA1 stratum radiatum to determine the: (1) frequency of multiple (MSB) vs. single (SSB) synapse boutons; (2) number of synaptic contacts per MSB; (3) bouton volume and surface area; and (4) types of spines in synaptic contact with MSBs and SSBs in ovariectomized, estradiol-treated animals (OVX + E) versus ovariectomized oil-treated controls (OVX + O). Quantitative analysis of this tissue revealed that, in OVX + E animals, 45.0% of presynaptic boutons form multiple synaptic contacts with dendritic spines compared to 27.3% in controls (P < 0.01); the average number of synapses per dendritic spines compared to 27.3% in controls (P < 0.01); the average number of synapses per MSB was 2.7 in OVX + E animals compared to 2.3 in controls (P < 0.05). This represents a 25.5% increase in the number of synapses formed by a given number of presynaptic boutons in estradiol-treated animals (P < 0.01) which largely accounts for the previously observed estradiol-induced increase in axospinous synapse density. There was no treatment effect on bouton size; however, because MSBs are larger than SSBs, the increased frequency of MSBs in estradiol-treated tissue results in a trend toward an estradiol-induced increase in average bouton size. Additionally, MSBS were found to be more irregular in shape, i.e., significantly less spherical, than SSBs. Our results indicate that estradiol-induced dendritic spines form synapses primarily with preexisting boutons in stratum radiatum and that these boutons enlarge and change shape as they accommodate new synapses. Such findings suggest a relatively active role for dendrites in the process of adult synapse formation.

Oestrogens and the structural and functional plasticity of neurons: implications for memory, ageing and neurodegenerative processes.

Oestrogens have numerous effects on the brain, beginning during gestation and continuing on into adulthood. Many of these actions involve areas of the brain that are not primarily involved in reproduction, such as the basal forebrain, hippocampus, caudate putamen, midbrain raphe and brainstem locus coeruleus. This paper describes three actions of oestrogens that are especially relevant to brain mechanisms involved in memory processes and their alterations during ageing and neurodegenerative diseases: (1) the regulation of cholinergic neurons by oestradiol in the rat basal forebrain, involving induction of choline acetyltransferase and acetylcholinesterase according to a sexually dimorphic pattern; (2) the regulation of synaptogenesis in the CA1 region of the hippocampus by oestrogens and progestins during the four- to five-day oestrus cycle of the female rat. Formation of new excitatory synapses is induced by oestradiol and involves N-methyl-D-aspartate receptors; removal of these synapses involves intracellular progestin receptors; (3) sex differences in hippocampal structure, which may help to explain differences in the strategies that male and female rats use to solve spatial navigation problems. During the period of development when testosterone is elevated in the male, aromatase and oestrogen receptors are also elevated, making it likely that this pathway is involved in the masculinization of hippocampal structure.

Estradiol regulates hippocampal dendritic spine density via an N-methyl-D-aspartate receptor-dependent mechanism.

In the adult female rat, the densities of dendritic spines and synapses on hippocampal CA1 pyramidal cells are dependent upon the ovarian steroid estradiol; moreover, spine and synapse density fluctuate naturally as ovarian steroid levels vary across the estrous cycle. To determine whether the effects of estradiol on dendritic spine density require activation of specific neurotransmitter systems, we have treated animals concurrently with estradiol and one of four selective neurotransmitter receptor antagonists: MK 801, a noncompetitive NMDA receptor antagonist; CGP 43487, a competitive NMDA receptor antagonist; NBQX, an AMPA receptor antagonist; or scopolamine, a muscarinic receptor antagonist. Our results indicate that the effects of estradiol can be blocked by treatment with either of the NMDA receptor antagonists, but treatment with an AMPA or muscarinic receptor antagonist has no effect on spine density. Thus, we have concluded that estradiol exerts its effect on hippocampal dendritic spine density via a mechanism requiring activation specifically of NMDA receptors.

Estradiol and progesterone regulate neuronal structure and synaptic connectivity in adult as well as developing brain.

Until recently, it has been widely believed that the adult brain does not undergo changes in its structure, particularly in relation to the actions of circulating hormones. It has now become clear that estradiol and progesterone have important effects on adult brain structure and function. Single section Golgi silver staining and electron microscopy have been used to analyze numbers of spines on dendrites and to count synapses on dendritic spines. In the adult female rat brain, we find that dendrites of neurons in the ventromedial hypothalamus and CA1 region of the hippocampus sprout increased numbers of spines on dendrites and then lose them during the 4- or 5-day estrous cycle. Increased spine numbers are accompanied by increased numbers of synapses on spines. In the hippocampus, the loss of spines and spine synapses occurs during a 24-h period between the time of maximum sexual receptivity on the day of proestrus and the next day, the day of estrus. This loss is not due solely to the decline in estradiol; however, giving progesterone speeds up the decline, and administering the antiprogestin, Ru486, on proestrus blocks the natural decline of synapse density. The changes of synaptic density in the hypothalamus are responsible, at least in part, for the cyclicity of sexual behavior, whereas the cyclicity of synapses in the hippocampus may subserve functions related to spatial learning and memory. In human subjects, cyclic fluctuations in gonadal hormones are associated with cyclic changes in performance on a variety of cognitive and motor tasks.

Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat.

We have previously shown that the density of dendritic spines on hippocampal CA1 pyramidal cells is dependent on circulating estradiol and progesterone and fluctuates naturally during the 5 day estrous cycle in the adult rat. To date, however, no detailed characterization of the roles that these hormones play in regulation of spine density has been made. In order to determine the time courses and extent of the effects of estradiol and progesterone on dendritic spine density, we have analyzed the density of dendritic spines on the lateral branches of the apical dendritic tree of Golgi-impregnated CA1 hippocampal pyramidal cells in several experiments. In summary, our findings included the following: (1) Following ovariectomy, circulating estradiol is undetectable within 24 hours; however, spine density decreases gradually over a 6 day period. (2) Spine density does not decrease any further up to 40 days following ovariectomy. (3) Treatment with estradiol alone can reverse the ovariectomy-induced decrease in spine density. (4) Spine density begins to increase within 24 hours following estradiol benzoate injection in an ovariectomized animal, peaks at 2 and 3 days, then gradually decreases over the next 7 day period. (5) Although free estradiol is metabolized more rapidly than estradiol benzoate, there is no difference in the rate of decrease in spine density following injection of either form. (6) Progesterone has a biphasic effect on spine density in that progesterone treatment following estradiol initially increases spine density for a period of 2 to 6 hours but then results in a much sharper decrease than is observed following estradiol alone. By 18 hours following progesterone treatment, spine density is decreased nearly to 6 day ovariectomy values. (7) Treatment of intact rats with the progesterone receptor antagonist, RU 486, during the proestrus phase of the estrous cycle inhibits the proestrus to estrus drop in spine density. These findings account for both the gradual increase and rapid decrease in spine density which we have previously observed during the estrous cycle and indicate that progesterone in particular may be an important factor in the regulation of rapid morphologic changes which occur naturally in the adult brain.

Differentiation of newly born neurons and glia in the dentate gyrus of the adult rat.

In order to determine whether newly born cells in the dentate gyrus of the adult rat express the neuronal marker, neuron-specific enolase, or the glial marker, glial fibrillary acidic protein, we performed combined immunohistochemistry and autoradiography on brains from adult rats perfused at various times ranging from 1 h to four weeks following [3H]thymidine administration. Light-microscopic examination revealed a negligible number of [3H]thymidine-labeled cells showing neuron-specific enolase immunoreactivity during mitosis. However, by two weeks after [3H]thymidine administration, a significant increase in the density of [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells was detected. Three weeks following [3H]thymidine injection the majority of [3H]thymidine-labeled cells (> 70%) were immunoreactive for the neuronal marker. At the four-week time-point, [3H]thymidine-labeled neuron-specific enolase-immunoreactive cells were indistinguishable from neighboring granule cells. In contrast, glial fibrillary acidic protein immunoreactivity was observed in a small but significant number of [3H]thymidine cells at the 1-h time-point and the proportion of labeled cells that were immunoreactive for this cell marker did not increase with time. [3H]Thymidine-labeled cells that were immunoreactive for glial fibrillary acidic protein typically showed morphologic characteristics of radial glia at all time-points. At the 1-h time-point, the majority of [3H]thymidine-labeled cells were observed in the hilus (> 60%) with the remainder being located in the granule cell layer. However, with a four-week survival-time most [3H]thymidine-labeled cells (> 85%) were located in the granule cell layer. The majority of newly born cells in the adult dentate gyrus differentiate into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)